Trophoblast cells are unique because they do not express major histocompatibility complex (MHC) class II antigens under any known conditions. The absence of MHC class 11 antigens from the surface of trophoblast cells is thought to be critical for maintenance of the fetus during pregnancy. Thus, successful reproduction of mammals may require that MHC class II gene expression be stringently repressed in trophoblast cells. The regulation of class II antigen expression is mediated at the level of transcription in trophoblast cell lines from species with hemochorial placentas. Mutation analysis of the mouse IAalpha promoter resulted in the identification of a novel trophoblast-specific negative regulatory element (IAalphaNRE) that functions to repress class II transcription. Subsequent work demonstrated that human, mouse and rat trophoblast cell lines all lack the class II transactivator (CIITA), a transacting transcription factor that is essential for both constitutive and IFNgamma-inducible transcription in other cell types. Transfection of human and rat trophoblast cells with CIITA expression vectors resulted in cell surface class II antigen expression. Based on these data, my working hypothesis is that expression of the class II genes may be regulated by two complementary mechanism(s) in trophoblast cells: 1) repression of CIITA synthesis, and 2) the activity of negative regulatory elements in the promoters of class II genes. Furthermore, I propose that CIITA gene transcription may be repressed in trophoblast cells by cis-acting sequences that function only in trophoblast and early embryonic cells. The specific aims of this, my first application, are to: 1) identify the cis-acting DNA sequences and transacting factors required for repression of CIITA gene transcription in trophoblast and embryonal carcinoma (EC) cells; 2) determine the mechanism by which the IAalphaNREs function to repress class II gene transcription in trophoblast cells; and 3) examine the expression of the transcription factors that regulate CIITA gene transcription in fresh human trophoblast. These studies will provide the first step in understanding the biological consequences of trophoblastic expression of class II antigens to successful pregnancy.